Bioactive Molecules from Nutrients:
In addition to the nutritional value of many foods, certain fractions and products of digestive pathways possess the ability to influence physiological processes. Some of these “extranutritional” constituents are present in their active form in the whole nutriment, such as the immunoglobulins in mother's milk and colostrums, phytoestrogens found in soy-based foods, polyphenolic antioxidants from fruits and vitamins. Others are encrypted within nutrient molecules, and are released in an active form during digestion or food processing, for example antihypertensive peptides from lactoglobin [Kitts, D. D. (1999), Can. J. Physiol. Pharmacol. 72:4; 423-434].
Biological Activity in Milk Proteins:
Milk contains a wide variety of proteins that contribute to it's unique qualities. Some proteins, such as bile-salt stimulated lipase, amylase, beta-casein, lactoferrin, haptocorrin and alpha-antitrypsin assist in digestion and utilization of milk-derived nutrients. Other proteins, such as immunoglobulins, kappa-casein, lyzozyme, lactoferrin and lactalbumin may, in the intact or partially digested form, have immunomodulatory and antimicrobial activity. Casein, the predominant milk protein, has been traditionally defined as composed of three fractions, α, β and γ, according to their electrophoretic mobility [N. J. Hipp, et al. (1952), Dairy Sci., 35:272]. Today casein is defined according to the amino acid sequences of each of the subgroups αS1, αS2, β and κ [W. N. Engel et al. (1984), J. Dairy Sci. 67: 1599].
In the course of digestion, the casein proteins are subjected to proteolytic cleavage by acid proteases such as chymosin (rennin), trypsin and pepsin, producing shorter peptides and causing curdling and calcium sequestration by the resultant protein fragments. A few studies with milk compounds demonstrated casein-related bacteriocidal activity. U.S. Pat. No. 3,764,670 discloses proteolytic casein digests possessing antibiotic properties against microorganisms. Israel Patent No. 42863 describes a casein-derived peptide consisting of 23 amino acids of the N-terminus of casein, possessing anti-bacterial activity. Shimizu et al. describe a short N-terminal fragment derived from αS1 casein peptic hydrolyzate having emulsifying properties, suggesting that this might be somehow useful to the food industry (Shimizu, et al. J. of Food Science, 1984;49: 1117-20). The authors investigated the amino acid composition of the fragment, it's in-vitro emulsifying activity, and noted that it resembled a 23 amino acid long N-terminal fragment of αS-1, concluding that the fragments were identical. However, no proof of identity was provided, and no biological activity was investigated.
In another study, Chabance et al. (Biochimie 1998;80:155-65) detected the presence of casein-derived peptides and peptide fragments in the stomachs and blood of humans after ingestion of yoghurt and milk. The authors reported the presence of fragments of bioactive κ-casein (caseinoglycopeptide) and an N-terminal fragment of αS-1 casein having antibacterial activity, in the blood following digestion. They concluded that the passage of these peptides, unaltered, into the plasma suggests a common, transport pathway for their duodenal absorption. No activity of the peptide fragments was demonstrated.
Lahov and Regelson describe a brief (30 minutes) chymosin digest of whole, acid-precipitated bovine and human casein, to yield a fraction enriched in an αS-1 casein N-terminal peptide (Lahov and Regelson, Fd Chem Toxic 1996;34:131-45), essentially duplicating the teachings of U.S. Pat. No. 3,764,670 to Katzir-Katchalsky et al. The chymosin digest was then precipitated with TCA, and characterized by centifugal analysis and short column equilibrium methods. The authors report an N-terminal αS-1 casein peptide fragment, similar to the anti-bacterial “isracidin” reported by Katzir-Katchalsky et al. However, the veracity of the author's claims to purification to homogeneity are questionable, considering the repeated detection of mixture of peptides reported in detailed studies of chymosin digest of casein employing sensitive analytical techniques (see, for example, Carles et al., FEBS Lett. 1985;115:282-6; McSweeney et al., J. Dairy Res., 1993;60:401-12, and Yvon, et al. Int. J. Pept. Prot Res, 1989;34:166-76).
In addition, other physiologically active properties, such as opioid and growth factor-like activities have been proposed for casein or its derivatives [Kitts, D. D., (1999), ibid.].
Immune modulating activity has also been observed in casein peptides. Coste et al. [Coste et al. (1992), Immun. Lett. 33: 41-46)] observed enhancement of rat lymphocyte proliferation following treatment with a peptide derived from the C-terminus of β casein. U.S. Pat. Nos. 5,506,209, 5,538,952 and 5,707,968, all to Mukerji et al. teach the administration of human β-casein, recombinant human β-casein, and hydrolysates of both, in a liquid enteral formula, for treating respiratory syncytial virus, otitis media, H. influenza and other infections in infants. Bovine β-casein was tested, but found to lack significant inhibitory activity, leading the authors to conclude that “β-casein from human milk has different bioactivity compared to bovine β-casein”.
U.S. Pat. Nos. 5,147,853 and 5,344,820 to Dosaka, et al. teach the administration of a sialic-acid conjugated κ-casein and κ-casein-derived glyco-macropeptide (GMP) from cow's milk for prevention of bacterial and viral infections in vitro and in vivo in rats. U.S. Pat. No. 5,330,975 to Isoda, et al. teaches the use of sialic-acid binding κ-casein and κ-casein peptides for the neutralization of bacterial endotoxins, such as cholera toxin. Similarly, U.S. Pat. No. 5,712,250 to Mukerji, et al., and U.S. Pat. No. 5,968,901 to Andersson, et al., teach the use of human κ-casein, but not bovine κ-casein, for the prevention of bacterial and H. influenza infection. However, these casein compositions taught in the prior art are relatively crude, even following gross fractionation, and none of these studies have determined the specific sequences in these casein peptides which confer their “extranutritional” properties.
Recent studies have detected a correlation between the consumption of the A1 β-casein fraction of bovine milk and Ischemic Heart Disease (IHD) in many Western countries (see, for example, M. Laugesen, NZ Med J. 2003;116:U295), leading to development of A1 β-casein-free milk (U.S. Pat. No. 6,570,060 to McLachlan).
Hematopoiesis in Cancer Therapy:
Following high-dose chemotherapy, especially following myeloablative doses of chemoradiotherapy supported by autologous bone marrow or peripheral blood stem cell transplantation (ASCT) or allogeneic bone marrow transplantation (BMT), patients are at high risk due to pancytopenia. Granulocytopenia may lead to development of serious, occasionally fatal infectious complications from common bacterial, viral, fungal and parasitic agents in the immediate post transplant period. Similarly, thrombocytopenia frequently results in bleeding tendency and occasionally, in long lasting platelet dependence. Whenever resistance to platelets develops, bleeding episodes can be life threatening and hemorrhagic complications are frequently lethal. The risk due to granulocytopenia can be partially overcome by supportive measures and most effectively by administration of recombinant human cytokines that can enhance reconstitution of granulocytes, particularly granulocyte colony stimulation factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). These agents are extremely expensive (approximately $200-400/day/patient) and infrequently cause side effects due to hypersensitivity reactions, fever, bone pain and occasionally vascular leak syndromes, including pericarditis and pleuritis. Some of the side effects may be due to other cytokines that may be intrinsically released by these hematopoietic growth factors. Moreover, the use of these hematopoietic growth factors may be prohibitive in patients with tumor cells bearing G-CSF or GM-CSF receptors such as in acute and chronic myeloid leukemias and in myelodysplastic syndromes. Whereas major progress in treating patients at risk of pancytopenia has been achieved from the use of hematopoietic cytokines, no progress has been made in the treatment of thrombocytopenia. Following high dose chemotherapy and especially following ASCT, patients are at risk for thrombocytopenia which may last for many months even up to 3 years and some thromboctyopenic patients may never recover. Many patients previously treated with multiple blood products become platelet resistant and hence thrombocytopenia may be impossible to overcome, even transiently, despite intensive and frequent platelet transfusions from a single donor. Resistance to platelets and protracted thrombocytopenia represent a common cause of death at ASCT centers worldwide.
Currently, several new recombinant cytokines such as recombinant human interleukin-3 (rhIL3) and recombinant human interleukin-6 (rhIL6) are being investigated as potential agents for enhancing megakaryocytopoiesis and platelet reconstitution. Unfortunately, preliminary clinical trials showed that although rhIL3 and rhIL6 may enhance platelet reconstitution, such effects are by no means dramatic and may take considerable time.
Clearly, protracted thrombocytopenia represents a major problem in clinical Bone Marrow Transplant centers today, for which no satisfactory solution has yet been found.
There is thus a widely recognized need for, and it would be highly advantageous to have a safe, inexpensive, rapidly effective and well-defined stimulator of hematopoiesis, and specifically megakaryocytopoiesis, devoid of the above limitations.
Thrombopoetin (TPO) in Regulation of Hematopoiesis and Platelet Function:
TPO appears to be the major regulator of platelet production in vivo, although increase in the kidney- and liver-derived growth factor in platelet deficiencies is not caused by adaptation of TPO biosynthesis in these organs. Rather, a “feed-back loop” seems to exist in which the number of circulating platelets determines how much of the circulating TPO is available to the bone marrow for platelet production. In addition, it has been demonstrated that TPO is an early acting cytokine with important multilineage effects: TPO alone, or in combination with other early acting cytokines, can (i) promote viability and suppress apoptosis in progenitor cells; (ii) regulate hematopoietic stem cell production and function; (iii) trigger cell division of dormant multipotent cells; (iv) induce multilineage differentiation and (v) enhance formation of multilineage colonies containing granulocytes, erythrocytes, macrophages, and megakaryocytes (MK, CFU-GEMM). Moreover, TPO stimulates the production of more limited progenitors for granulocyte/monocyte, megakaryocyte and erythroid colonies, and stimulates adhesion of primitive human bone marrow and megakaryocytic cells to fibronectin and fibrinogen. Thus, TPO is an important cytokine for clinical hematologists/transplanters: for the mobilization, amplification and ex vivo expansion of stem cells and committed precursor cells for autologous and allogeneic transplantation [von dem Borne, A. E. G. Kr., et al., (1998) Thrombopoietin: it's role in platelet disorders and as a new drug in clinical medicine. In Bailliers Clin. Hematol. Jun.:11(2), 427-45].
In addition to TPO effects in hematopoiesis, this potent growth factor primes platelets for various agonists and modulates platelet-extracellular matrix interactions. Although it does not itself cause platelet aggregation, TPO upregulates ADP-induced aggregation, especially on the second wave of aggregation, upregulates granule (ADP, ATP, serotonin, etc.) release and production of thromboxane B2, increases platelet attachment to collagen and potentiates shear-induced platelet aggregation. TPO also stimulates PMN activation, inducing IL-8 release and priming oxygen metabolite production, likely enhancing antimicrobial defense.
Clinical studies suggest TPO's value in understanding and treating a variety of hematological conditions. In patients with idiopathic aplastic anemia (AA), elevated TPO levels persist even in remission following Immunosupressive therapy, indicating a hematopoietic defect. TPO is elevated in other forms of aplastic thrombocytopenia as well, but not in conditions of increased platelet destruction. Apparently, the reactive increase in TPO production is insufficient in cases of destructive thrombocytopenia. Thus, TPO is not only a therapeutic option for aplastic, but also for destructive thrombocytopenia.
Thrombopoietic agents are of great clinical interest, for prevention and/or treatment of pathological or treatment-induced thrombocytopenia, and as a substitute for platelet transfusions. Of the cytokines evaluated, all but the marginally potent IL-11 have been deemed unacceptable for clinical use. TPO is widely believed to become the cytokine of choice for throbocytopenia treatment. Recombinant human TPO (Genentech) has recently become available, enabling accurate pharmacokinetic determinations and clinical trials. Thus, TPO's potential applications encompass the realms of supportive care (post chemo/radio-therapy, bone marrow and stem cell transplantation), hematological disease (AA, myelodysplasia, congenital and acquired thrombocytopenia), liver diseases, transfusion (expansion, harvest, mobilization and storage of platelets) and surgery (including liver transplantation). Of particular interest is the potential use of TPO/EPO/G-CSF cocktail for myelodysplasia, G-CSF and TPO combination for peripheral stem cell mobilization and TPO in harvesting CD 34+ cells and ex vivo expansion of megakaryocytes for superior platelet reconstitution. Recombinant human G-CSF is also available (Filgrastim, Amgen Inc., USA). However, similar to other hematopoietic agents under consideration for clinical application, TPO and G-CSF are costly and potentially antigenic at therapeutically effective levels. Thus, it would be advantageous to have a safe, inexpensive and readily available stimulator of thrombopoiesis and granulocytopoiesis capable of augmenting TPO and G-CSF activity.
SARS:
The worldwide outbreak of severe acute respiratory syndrome (SARS), and reported SARS-related deaths in more than 25 countries in the spring of 2003 has focused attention on the suspected infective agent, the SARS-CoV coronavirus (Rota et al., Sciencexpress, 1 May 2003). Evidence of SARS-CoV infection has been documented in SARS patients throughout the world, SARS-CoV infection has been detected in respiratory specimens, and convalescent-phase serum from SARS patients contains anti-SARS antibodies. Presently, no therapies have been identified for the prevention or treatment of SARS-CoV infection.
In the absence of effective vaccines or drugs, the current SARS epidemic threatens to reach devastating proportions, similar to epidemics of other infectious diseases spread by respiratory route such as the influenza epidemic of 1918 and measles epidemics. As has been emphatically stated by many health officials, the key to controlling epidemics is the blockage of transmission of infection. Thus, in addition to much needed public health measures, the development of methods for prevention and/or treatment of SARS is of foremost importance.
The αS-1 , κ- , and ,β-fraction of casein:
The αS1 fraction of casein can be obtained from milk proteins by various methods [D. G. Schmidth and T. A. J. Paynes (1963), Biochim., Biophys. Acta, 78:492; M. P. Thompson and C. A. Kiddy (1964), J. Dairy Sci., 47:626; J. C. Mercier, et al. (1968), Bull. Soc. Chim. Biol. 50:521], and the complete amino acid sequence of the αS1 fraction of casein was determined by J. C. Mercier et al. (1971) (Eur. J. Biochem. 23:41). The genomic and coding sequences of bovine αS1 fraction of casein have also been cloned and sequenced employing recombinant DNA techniques [D. Koczan, et al. (1991), Nucl. Acids Res. 19(20): 5591; McKnight, R. A., et al. (1989), J. Dairy Sci. 72:2464-73]. Proteolytic cleavage and identification of N-terminal fragments from the αS1 fraction of casein has been documented [J. C. Mercier, et al. (1970), Eur. J. Biochem. 16:439; P. L. H. McSweeney et al. (1993), J. Dairy Res., 60:401], as has the intestinal absorption and appearance of this fragment in mammalian plasma following ingestion of whole milk proteins [Fiat, A. M., et al. (1998) Biochimie, 80(2):2155-65]. Meisel, H. and Bockelmann, W. [(1999), Antonie Van Leeuwenhoek, 76:207-15] detected amino acid sequences of immunopeptides, casokinins and casomorphins in peptides liberated by lactic acid bacteria digests of α and β casein fractions. Of particular interest is the anti-aggregating and thrombolytic activity demonstrated for C-terminal portions of the α- and κ-casein fractions [Chabance, B. et al. (1997), Biochem. Mol. Biol. Int. 42(1) 77-84; Fiat A M. et al. (1993), J. Dairy Sci. 76(1): 301-310].
The coding sequences for bovine αS2-, β- and κ-casein have also been cloned (Groenen et al., Gene 1993; 123:187-93, Stewart, et al., Mol. Biol. Evol. 1987:4:231-41, and Stewart, et al., Nucl. Acids Res. 1984;12:3895-907). The αS2-casein coding sequence has numerous Alu-like retroposon sequences, and, although the gene is organized similarly to the αS1-casein gene, sequence analysis indicates that it is more closely related to the β-casein-encoding gene. β-casein is characterized by numerous clusters of serine residues, which, when phosphorylated, can interact with and sequester calcium phosphate (Stewart et al., Mol Biol Evol. 1987;4:231-43). κ-casein is a smaller polypeptide, the amino acid and nucleotide sequence of which (Alexander et al., Eu. J. Biochem. 1988;178:395-401) indicates that it is evolutionarily unrelated to the calcium-sensitive casein gene family. In the gut, κ-casein is split into an insoluble peptide (para-kappa casein) and a soluble hydrophilic glycopeptide (caseinomacropeptide), which has been shown to be active in efficiency of digestion, prevention of neonate hypersensitivity to ingested proteins, and inhibition of gastric bacterial pathogens (Malkoski, et al., Antimicrob Agents Chemother, 2001;45:2309-15).
Previous studies have documented potential bioactive peptides encrypted in the N-terminal αS-1 casein, the αS2-casein, β-casein and in the κ-casein amino acid sequences, but no mention was made of use of these protein fragments, specific sequences or defined synthetic peptides to enhance hematopoiesis, prevent viral infection or modulate the development of autoimmune diseases.
The present invention successfully addresses the shortcomings of the presently known art by providing peptides for the treatment of human disease, which peptides are derived from the N terminus portion of αS1 casein and posses no detectable toxicity and high therapeutic efficacy in a myriad of pathological indications.